Image analysis

ABSTRACT

Provided herein is technology relating to analysis of images and particularly, but not exclusively, to methods and systems for determining the area and/or volume of a region of interest using optical coherence tomography data. Some embodiments provide for determining the area and/or volume of a lesion in retinal tissue using three-dimensional optical coherence tomography data and a two-dimensional optical coherence tomography fundus image.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit U.S. Provisional Patent ApplicationNo. 62/269,601 filed Dec. 18, 2015 and U.S. Provisional PatentApplication No. 62/138,485, filed Mar. 26, 2015, each of which isincorporated by reference in its entirety.

FIELD

Provided herein is technology relating to analysis of images andparticularly, but not exclusively, to methods and systems fordetermining the area and/or the volume of a region of interest usingoptical coherence tomography data.

BACKGROUND

Clinicians and researchers increasingly use image data representingbiological tissues, e.g., to identify and diagnose tissue anomalies andpathologies. Imaging using optical coherence tomography (OCT) is animaging method that produces cross-sectional images of tissue morphology(see, e.g., Pieroni et al. (2006) “Ultrahigh resolution opticalcoherence tomography in non-exclusive age related macular degeneration”Br J Ophthalmol 90(2): 191-7).

In particular, OCT provides excellent visualization of retinal tissueabnormalities using cross-sectional pseudo-color or grayscale images ofthe tissue reflectivity. However, quantifying the size (e.g.,determining the lateral extent, area, and/or volume) of localizedretinal tissue abnormalities is not trivial. For example, determiningthe en face area of a retinal abnormality in OCT data alone (e.g.,without reference to a fundus image) involves a user identifying theborder of the retinal abnormality in each of many OCT cross-sectionalimages. This is a very laborious process and is not accurate when theabnormality has multiple loci, a complex or irregular shape, or when theextent of the abnormalities increases laterally. Further, OCTsegmentation is typically based on known anatomical tissue layers (e.g.,in a normal subject) and thus is not necessarily based on the border ofthe abnormality.

For example, extant technologies for evaluating a volumetric size (e.g.,the volume of the retinal cystoid space) often include steps wherein aclinician views an OCT image and manually segments various anatomicallayers (e.g., the inner limiting membrane (ILM) and the retinal pigmentepithelium (RPE)) shown in the image. Then, software performs avolumetric calculation, e.g., by a trapezoidal integration of multipleframes of the thickness between the anatomical layers. Using such atechnique, while the resultant value includes the volume of theabnormality under examination (e.g., the cystoid space), it alsoincludes the volume of the neighboring (e.g., healthy) tissue that maynot be relevant to the calculation related to the abnormality. Further,some extant technologies are associated with measuring the variance ofthe volume of a tissue from the development of the abnormality bycomparison to a normative database. These measurements, however, areconfined to the fixed regions and the fixed segmented layers that haveto be measured to generate the normative data. In addition, thesetechnologies are limited in that such measurements are not typicallymade and thus appropriate data are not available.

In addition, quantitative analysis of OCT data has been used in thediagnosis and treatment of macular degeneration (e.g., wet and dryage-related macular degeneration (AMD)). For example, retinal thicknessmeasurements have been used for some patients to monitor theeffectiveness of treatment with anti-vascular endothelial growth factor(VEGF) agents (e.g., Ranibizumab marketed as, e.g., “Lucentis”;aflibercept marketed as, e.g., “Eylea”). Measurement of retinalthickness (e.g., measurement of a fixed 1-mm (e.g., 0.1 to 10 mm)diameter region centered on the macula) is used to estimate thesubretinal and/or the intraretinal fluid accumulation and is generallyaccepted by the FDA as an imaging endpoint. In addition, new combinationtherapies are available that target both VEGF and platelet-derivedgrowth factor (PDGF). While the anti-PDGF treatment is administered toreduce the size of the central neovascularization (CNV) complexassociated with macular degeneration, quantitative analysis of thistreatment in human clinical trials is presently limited. Severalpreliminary studies have attempted to quantify the lateral extent (e.g.,the area) of the CNV lesion complex using fluorescein angiography imagesbut have not quantified the area or volume of a CNV lesion based on OCTdata (see, e.g., Jaffe et al. (2015) “A phase 1 study of intravitreousE10030 in combination with Ranibizumab in neovascular AMD” Ophthalmology(Manuscript no. 2015-470, in press); Boyer (2009) “Combinationinhibition of PDGF and VEGF for treatment of neovsacular AMD”, ARVOabstract).

Area measurements of retina lesions also find use as anatomic endpoints,e.g., for monitoring and treating geographic atrophy (GA) of the retinapigment epithelium (RPE) in AMD. In particular, an increase in the areaof the GA region over time is considered a measure of diseaseprogression. In addition, a clinically important measure is theproximity of the boundary of the GA (as measured by loss of the RPEand/or disappearance of the external limiting membrane layer of theretina) to the retinal area for the center of vision, the fovea. Atreatment that slows the progression of GA (e.g., slows the increase ofthe area of the GA region as a function of time) and/or delays theinvasion of the GA region toward and/or into the fovea may preservevision and/or minimize the loss of vision. Numerous treatments for GAare under clinical investigation at this time and would benefit fromtechnologies that measure and/or monitor the size and/or change in sizeof a GA region. Current technologies for tracking GA progression in bothclinical care and clinical research are based on measuring the area of aGA region using en face imaging modalities such as, e.g., retinaphotographs, fundus autofluorescence, fluorescein angiograms, etc.Further, present OCT-based measurements of GA are based on voxelprojection images (see, e.g., Hu (2013) “Segmentation of GeographicAtrophy in Spectral-Domain Optical Coherence Tomography and FundusAutofluorescence Images” Invest Ophth Vis Sci 54: 8375-83; Yehoshua(2014) “Comparison of Geographic Atrophy Measurements from the OCTFundus Image and the Sub-RPE Slab Image” Ophthalmic Surg Lasers ImagingRetina 44: 127-32).

Thus, although OCT data are valuable to clinicians and researchers, theutility of OCT technologies would benefit from improved image analysisfor measuring the sizes of tissue anomalies and pathologies, e.g., bydirectly correlating eye microstructures using three-dimensional (e.g.,volumetric) metrics and two-dimensional (e.g., en face) display of data.

SUMMARY

The metric assessment (e.g., determination of one or more sizes (e.g.,distances, areas, volumes, etc.)) and tracking of localized tissueabnormalities provides a diagnostic tool, e.g., for the treatment ofsubjects. In particular, the technology described herein relates to amethod of pairing optical coherence tomography (OCT) three-dimensional(e.g., volume and/or cross-sectional) data with a two-dimensional image(e.g., a fundus image, a color photograph, infrared reflectance image,angiographic frame, integration of three-dimensional data, etc.) andusing the two-dimensional image and/or cross sectional data (e.g.,two-dimensional “slices” of three-dimensional data) as the primarysource to determine the extent (e.g., in linear (e.g., one-dimensional),two-dimensional, and/or three-dimensional space) of a tissueabnormality.

In various embodiments, the fundus image is or is not acquired togetherwith the OCT data during data acquisition. In some embodiments, thefundus image is registered with the OCT data (e.g., OCT image) post hoc.In some embodiments, the OCT data (e.g., OCT image) is registeredpixel-by-pixel with the fundus image. In some embodiments, the referencefundus image is an en face pixel display of OCT data.

In some embodiments, a user indicates a boundary of the region ofinterest (e.g., abnormality, lesion, CNV complex, etc.) on a displayshowing the fundus image and/or by examination of the OCT data, thusproviding a digital representation (e.g., a pixel representation) of theborder of the region of interest (e.g., abnormality, lesion, CNVcomplex, etc.). The digital representation of the region of interest(e.g., abnormality, lesion, CNV complex, etc.) is mapped to a digitalrepresentation (e.g., a pixel representation) of the OCTthree-dimensional data and the software determines the area and/orvolume of the region of interest (e.g., abnormality, lesion, CNVcomplex, etc.) from the fundus image and/or OCT data according to thetechnology provided herein.

Accordingly, the technology provided herein relates in some embodimentsto a method for determining the distance, length, or location; area;and/or volume of a region of interest of a biological tissue usingoptical coherence tomography (OCT). In some embodiments, the methodcomprises acquiring three-dimensional OCT data comprising at least afirst segment (e.g., an anterior segment) and a second segment (e.g., aposterior segment) defining the region of interest; acquiringtwo-dimensional image data (e.g., OCT fundus data, a photograph, etc.)comprising the region of interest; providing a boundary around theregion of interest in the two-dimensional image data, the boundaryenclosing an area A; calculating the volume v within the boundary ofarea A and between the first segment and the second segment; calculatingthe average thickness t between the first segment and the second segmentalong the boundary (e.g., along the perimeter of area A); andcalculating the volume V of the region of interest:V=v−(t×A)

The technology is not limited by the shape of the boundary. For example,in some embodiments the boundary is a circle, ellipse, polygon (e.g.,triangle, quadrilateral (e.g., square, rectangle, trapezoid,parallelogram, rhombus, etc.), etc.), or other shape, and in someembodiments the boundary is an irregular shape. In some embodiments, theboundary is any shape whose perimeter surrounds the region of interest(e.g., in a fundus image or other two-dimensional representation (e.g.,projection, integration, slice, cross-section, etc.) ofthree-dimensional data) and that has an area (e.g., an area that is thesame size or larger than the limits of the lesion and/or region ofinterest). Some embodiments comprise determining a greatest lineardistance across the region of interest in the two-dimensional OCT fundusdata. In some embodiments, the greatest linear distance is determined orprovided by a computer and in some embodiments the greatest lineardistance is determined or provided by a user. In some embodiments, alinear measurement provides a distance of a lesion boundary to ananatomic location in the retina (e.g., a distance to the fovea from thenearest edge of a lesion).

Particular embodiments relate to OCT data wherein the three-dimensionalOCT data is a three-dimensional OCT image and wherein thetwo-dimensional OCT fundus data is a two-dimensional image, e.g.,produced by integrating three-dimensional OCT data, or wherein thetwo-dimensional OCT fundus data is a photograph (e.g., a digital image).

In some preferred embodiments, a user draws the boundary around theregion of interest (e.g., by interacting with a computer to provide apixel representation of the boundary superimposed on the fundus image);in specific embodiments, a user draws the boundary around the region ofinterest using a computer input device (e.g., a cursor control device,e.g., a mouse, light pen, stylus, touch screen, trackball, trackpad,joystick, etc.). In some embodiments, automated image processing drawsthe boundary around the region of interest. For example, in someembodiments a user identifies points on an image defining the edge of aregion of interest and an automated method (e.g., a software method)connects the points to provide a continuous boundary encompassing theregion of interest. The automated image analysis algorithm analyses theimaging data and the location of the user-defined points to provide aboundary encompassing the region of interest (e.g., by using aninterpolation algorithm to define points between the user-defined pointsand connecting all points with a line encompassing the region ofinterest).

Embodiments of the technology are provided to provide an area and/or avolume of a region of interest (e.g., tissue abnormality, lesion, CNVcomplex, etc.). Embodiments of the technology calculate the area and/orvolume of the region of interest (e.g., abnormality, lesion, CNVcomplex, etc.) by identifying a region of the tissue that is normal.Accordingly, some embodiments comprise providing the boundary on normalbiological tissue such that the region of interest is circumscribed byan unaffected boundary region or through the use of a normative database(e.g., the perimeter of the boundary is provided on a region of theimage data corresponding to normal tissue). Some embodiments provide foroutputting the area and/or volume of the region of interest to a user(e.g., on a display, over a network, printed on paper, etc.).

In some embodiments, the region of interest is a lesion in thebiological tissue. In particular embodiments, the biological tissue isretinal tissue and the region of interest is a retinal lesion. Forexample in some embodiments the region of interest is a centralneovascularization (CNV) lesion complex, e.g., associated with maculardegeneration (e.g., wet macular degeneration, dry macular degeneration).In other embodiments, the region of interest is a defect of the retinapigment epithelium layer and/or the volume of a specific retina layeroverlying a region of interest (e.g., the outer nuclear layer of theretina over an area of RPE loss).

Additional methods relate to treating a subject having a tissueabnormality (e.g., abnormal tissue growth, CNV lesion, etc.). Thus, someembodiments provide a method of treating a subject having a tissueabnormality, the method comprising acquiring an area and/or a volume ofthe tissue abnormality according to a method provided herein andadministering a treatment to the subject based on the area and/or thevolume of the tissue abnormality. Additional embodiments relate to amethod of identifying that a treatment of a subject having a tissueabnormality is successful, the method comprising calculating a firstarea and/or a first volume of the tissue abnormality according to amethod as described herein; administering a treatment to the subject;calculating a second area and/or a second volume of the tissueabnormality according to a method described herein; and identifying thetreatment of the patient as effective when the second area and/or thesecond volume of the tissue abnormality is less than the first areaand/or the first volume of the tissue abnormality. For example, in someembodiments an effective treatment reduces the area and/or the volume ofthe tissue abnormality by 1% to 100% (e.g., from 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 to 100%).

Some embodiments relate to treating a subject and monitoring theprogress in treatment using a linear measurement, e.g., a measurementthat provides a distance of a lesion boundary to an anatomic location inthe retina (e.g., a distance to the fovea from the nearest edge of alesion). For example, in some embodiments an effective treatmentincreases the distance between the lesion and the anatomical feature(e.g., the fovea) by 1% to 100% (e.g., from 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 to 100%).

Accordingly, in some embodiments a first area and/or a first volume ofthe tissue abnormality is measured according to a method providedherein; a second area and/or a second volume of the tissue abnormalityis measured according to a method provided herein after the first areaand/or the first volume measurement; and a second area and/or a secondvolume that is less than the first area and/or first volume indicatesthat the treatment is effective. In some embodiments a first distance ofthe tissue abnormality from an anatomical feature is measured accordingto a method provided herein; a second distance of the tissue abnormalityfrom the anatomical feature is measured according to a method providedherein after the first distance measurement; and a second distance thatis greater than the first distance indicates that the treatment iseffective

Thus, in some embodiments in which the treatment is indicated aseffective, the treatment is modified (e.g., dose decreased),discontinued, changed, etc. In some embodiments, the distance, area,and/or volume measurements indicate that one treatment should replaceanother treatment. In some embodiments, the distance, area, and/orvolume measurements indicate that two or more treatments should becombined.

In some embodiments, the areas and/or volumes indicate that thetreatment is effective and that it should thus be maintained withoutmodification. Conversely, an increase in the second area and/or thesecond volume measurement relative to the first area and/or first volumemay indicate disease progression. In some embodiments, tissue loss(e.g., atrophy) is determined by an increasing negative value of thesecond measurement (e.g., the difference between the first area and/orfirst volume and subsequent second areas and/or second volumesincreases). In some embodiments, a reference region is selected in aplurality of images to provide a common reference point for comparingand/or aligning (e.g., registering) the plurality of images. Forexample, in some embodiments, images from different time points areregistered with each other (e.g., using anatomical (e.g., tissue)landmarks such as retinal blood vessels, the optic nerve head, etc.).

In some embodiments, a subject is tested to assess the presence, theabsence, or the level of a tissue abnormality, e.g., by acquiring anarea and/or a volume of the tissue abnormality according to a methodprovided herein, and thereafter the subject is treated based on theoutcome of the test. In some embodiments, a subject is tested, treated,and then tested again to monitor the response to therapy. In someembodiments, cycles of testing and treatment may occur withoutlimitation to the pattern of testing and treating (e.g., test/treat,test/treat/test, test/treat/test/treat, test/treat/test/treat/test,treat/test/treat, test/treat/treat/test/treat/treat, etc), theperiodicity, or the duration of the interval between each testing andtreatment phase.

The technology provided herein provides several advantages relative toexisting OCT metric analysis. For example, existing analysis softwarefor processing OCT data scans uses a sampled area that is fixed (e.g., agridded circle having a 6-mm diameter and centered on the fovea) andsegments the inner and outer limits of the tissue layers within thatcircle. The area of the total tissue and the volume of the total tissuearea are calculated by integrating all points within the fixed referencecircle. However, the actual area and/or volume of the localized lesion(e.g., tissue and swelling from a choroidal neovascular membrane) may beonly a small fraction of the total sampled area and, consequently, avery large area of healthy tissue may be included in the measured areaand/or volume of the grid using the existing technology. Thus, a smallrelative change in the 6-mm circle volume (e.g., 5%) may represent avery large relative change in the volume of the localized lesion (e.g.,a decrease of 80%). In contrast, the technology described hereinprovides a more accurate and sensitive biomarker for measuring therelative metric (e.g., area or volume) change of localized lesions indata for analysis.

Additional embodiments provide a system for determining the area and/orvolume of a region of interest of a biological tissue, the systemcomprising an OCT apparatus and software to calculate an area and/orvolume of the region of interest according to the methods describedherein. Further embodiments comprise a component configured to displaythree-dimensional OCT data and two-dimensional fundus data to a user.Yet additional embodiments of systems comprise a component configured toaccept input from a user to provide the boundary around the region ofinterest in the two-dimensional OCT fundus data. Particular embodimentsprovide a component to output the area and/or volume of the region ofinterest, e.g., to a user.

Some embodiments provide a method for determining the area and/or volumeof a retinal lesion, the method comprising acquiring SD-OCT data (e.g.,three-dimensional SD-OCT data); displaying a fundus image of the SD-OCTdata on a display (e.g., produced from the three-dimensional SD-OCTdata; acquired simultaneously with the three-dimensional SD-OCT data;and/or acquired at a different time than the three-dimensional SD-OCTdata); and providing a boundary around a region of interest by acquiringuser input from a user who interacts with the displayed two-dimensionalfundus image using an input device, the boundary enclosing an area A.

In particular embodiments provided herein is a method for determiningthe area and/or volume of a retinal lesion, the method comprisingproviding a three-dimensional SD-OCT image comprising a first retinalsegment and a second retinal segment comprising a retinal lesion;providing a two-dimensional SD-OCT fundus image comprising the retinallesion; determining a greatest linear distance across the retinal lesionin the two-dimensional fundus image; circumscribing a circle around theretinal lesion in the two-dimensional fundus image, the circle having adiameter d greater than or equal to the greatest linear distance acrossthe retinal lesion in the two-dimensional fundus image; calculating theaverage thickness t between the first retinal segment and the secondretinal segment along the perimeter of the circle; calculating thevolume v within the circle and between the first segment and secondsegment; and calculating the volume V of the retinal lesion:V=v−(t×Π×(d/2)²)

In particular embodiments provided herein is a method for determiningthe area and/or volume of central neovascularization (CNV) lesion, e.g.,associated with macular degeneration, the method comprising providing athree-dimensional SD-OCT image comprising a first retinal segment and asecond retinal segment comprising a CNV lesion; providing atwo-dimensional SD-OCT fundus image comprising the CNV lesion;determining a greatest linear distance across the CNV lesion in thetwo-dimensional fundus image; circumscribing a circle around the CNVlesion in the two-dimensional fundus image, the circle having a diameterd greater than or equal to the greatest linear distance across the CNVlesion in the two-dimensional fundus image; calculating the averagethickness t between the first retinal segment and the second retinalsegment along the perimeter of the circle; calculating the volume vwithin the circle and between the first segment and second segment; andcalculating the volume V of the CNV lesion;V=v−(t×Π×(d/2)²)

Some further embodiments provide a method for determining the areaand/or volume of a CNV lesion, the method comprising acquiring SD-OCTdata (e.g., three-dimensional SD-OCT data); displaying a fundus image ofthe SD-OCT data on a display (e.g., produced from the three-dimensionalSD-OCT data; acquired simultaneously with the three-dimensional SD-OCTdata; and/or acquired at a different time than the three-dimensionalSD-OCT data); and providing a boundary around a region of interest byacquiring user input from a user who interacts with the displayedtwo-dimensional fundus image using an input device, the boundaryenclosing an area A.

In some embodiments, the method further comprises segmenting the SD-OCTdata, e.g., to provide at least a first segment and a second segment. Insome embodiments, the segments correspond to anatomical features (e.g.,tissues, tissue layers, etc.) and in some embodiments the segments donot necessarily correspond to histological, biological, and/oranatomical features (e.g., the segments are appropriate for analysis ofthe region of interest as provided by the technology herein and notnecessarily with respect to histological, biological, and/or anatomicalfeatures).

In yet additional embodiments, the method comprises calculating thevolume v within the boundary and between the first segment and thesecond segment; calculating the average thickness t between the firstsegment and the second segment along the boundary; and/or calculatingthe volume of the region of interest V=v−(t×A).

In some embodiments, the method comprises identifying regions and/orboundaries of RPE loss from OCT B scans in eyes with geographic atrophyof the retina pigment epithelium. In some embodiments, identifying theregions and/or boundaries of RPE loss comprises registering thelocations of the regions and/or boundaries in the OCT scans with thecorresponding locations in the en face fundus image. In someembodiments, the area A is then calculated using segmentation (e.g.,manual segmentation by the user and/or automated segmentation) of theboundary annotated to the en face image. Accordingly, embodimentsprovide that images acquired at multiple time points are assessedaccording to the same methods and differences in lesion boundariesand/or lesion areas in a plurality of images are used to assess changesin boundaries, areas, shapes, etc., e.g., to provide an assessment ofdisease progression, treatment, etc.

In some particular embodiments, the input device is a touchscreen—thatis, the user interacts with the data displayed as an image on atouchscreen and the user provides commands and indicates the boundary ofthe region of interest by providing input using the touchscreen (e.g.,by drawing with a finger, stylus, etc. on the touchscreen and/or usinganother input device). In some embodiments, the methods further comprisedisplaying the boundary on the display (e.g., superimposed on the SD-OCTimage). For example, in some embodiments, a distance (e.g., a distanced, a distance from a lesion (e.g., a lesion boundary) to an anatomicalfeature, etc.) is displayed on the display, an area A of the volume ofinterest is displayed on the display, a volume of the region of interestV is displayed on the display, and/or the average thickness t isdisplayed on the display. In some embodiments, as the boundary ischanged by user interaction, the methods comprise updating the volume ofthe region of interest V on the display and updating the averagethickness t on the display as the boundary changes.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is a schematic drawing showing an exemplary coordinate system inrelation to an OCT apparatus (“OCT”) and a sample (“sample”) imaged bythe OCT apparatus.

FIG. 2A is a schematic drawing showing a cross-section image of a regionof interest in a segmented image of a tissue sample (lower panel) andthe associated fundus image showing the region of interest (upperpanel). The lower panel shows a first segment and a second segment as anupper black line and a lower black line. The upward protrusion in theupper black line represents a growth abnormality, lesion, etc., or otherregion of interest of a sample.

FIG. 2B is a schematic drawing showing a boundary provided around theregion of interest in the fundus image (upper panel). The region ofinterest has a greatest linear dimension g and, in some embodiments, theboundary is a circle having a diameter d. The boundary defines an area A(upper panel, grey region). Extension of the boundary through the imageto cross the segments defines a volume v enclosed by the first segment,second segment, and extended boundary (lower panel, grey region).

FIG. 2C is a schematic drawing showing a volume n (white region) incross-section, calculated from the area A and the distance t (e.g., anaverage thickness between the first segment and the second segmentcalculated around the perimeter of the boundary) as described herein.The volume V of the region of interest is shown in the lower panel ingrey.

FIG. 2D is a schematic showing embodiments of the technology comprisingproviding a circular boundary having an area A (FIG. 2D-1) and providingan irregularly shaped boundary having an area A (FIG. 2D-3). FIG. 2D-2and FIG. 2D-4 show a volume n defined by the boundary extended throughthe sample over distance t. The volume n is the product of A and t.

FIG. 3A shows a fundus image and FIG. 3B shows an associated view of OCTdata. The long horizontal white line in the fundus image of FIG. 3Amarks the plane view of the OCT data displayed in FIG. 3B. The locationof the vertical white tick mark on the fundus image (FIG. 3A) iscorrelated to the location of the two vertical white tick marks on theOCT image (FIG. 3B).

FIG. 4A shows a fundus image and FIG. 4B shows an associated view of OCTdata obtained by imaging the retina of a subject. The long horizontalwhite line in the fundus image of FIG. 4A marks the plane view of theOCT data displayed in FIG. 4B. The location of the vertical white tickmark on the fundus image (FIG. 4A) is correlated to the location of thetwo vertical white tick marks on the OCT image (FIG. 4B). The region ofinterest encompassing a CNV lesion is outlined in white on FIG. 4A.

FIG. 5A shows a fundus image and FIG. 5B shows an associated view of OCTdata obtained at another (e.g., later) time point by imaging the samearea of the retina of the same subject from whom the image data in FIG.4A and FIG. 4B were obtained. The long horizontal white line in thefundus image of FIG. 5A marks the plane view of the OCT data displayedin FIG. 5B. The location of the vertical white tick mark on the fundusimage (FIG. 5A) is correlated to the location of the two vertical whitetick marks on the OCT image (FIG. 5B). The region of interestencompassing a CNV lesion is outlined in white on FIG. 5A.

FIG. 6A shows a fundus image (left panel) and an associated view of OCTdata (right panel) obtained by imaging the retina of a patient with AMDand GA. The horizontal white line on the fundus image corresponds to theOCT B scan shown. The white tick mark shows the corresponding locationbetween the OCT image and the fundus image at the boundary betweenaffected retina and RPE and relatively intact RPE/retina. FIG. 6B showsfundus image (left panel) and the OCT image (right panel) from the samepatient and visit whereby the boundaries of the atrophy have beenidentified on the OCT B scans and the corresponding locations are markedon the fundus image. The superior portion of the lesion has beenanalyzed in this image and the inferior portion has not yet beenanalyzed. FIG. 6C shows that the analysis has been completed and thearea of the GA has been completely circumscribed to define the regionwhereby the area A has been calculated.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

Provided herein is technology relating to analysis of images andparticularly, but not exclusively, to methods and systems fordetermining the volume of a region of interest using optical coherencetomography data.

In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein. Thesection headings used herein are for organizational purposes only andare not to be construed as limiting the described subject matter in anyway.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the invention may be readilycombined, without departing from the scope or spirit of the invention.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

As used herein, “optical coherence tomography” or “OCT” refers to amedical imaging technique that uses light to capturemicrometer-resolution, three-dimensional images from within opticalscattering media (e.g., biological tissue). Optical coherence tomographyis based on low-coherence interferometry, typically employingnear-infrared light. The use of relatively long wavelength light allowsit to penetrate into the scattering medium.

As used herein, an axis extending from an OCT apparatus and the sample(e.g., tissue) under examination is the z-axis. Planes normal to thez-axis are x-y planes. See, e.g., FIG. 1.

As used herein, an “A-scan” is an amplitude modulation scan thatprovides one-dimensional information in the direction of the z-axis,e.g., an axial depth scan. For example, in some embodiments an A-scan isused to determine the length of a tissue, tissue segment, tissuefeature, etc. in the direction of (or substantially, essentially, orapproximately along) the z-axis or to determine the location of a tissuesegment or tissue feature along a path in the direction of the z-axis.See, e.g., FIG. 1.

As used herein, a “B-scan” is a two-dimensional, cross-sectional or“profile” view of the sample under examination, e.g., a two-dimensionalscan in the x-z or y-z planes. The two-dimensional cross-sectionalB-scan may be produced by laterally combining a series of axial depthA-scans. See, e.g., FIG. 1.

As used herein, a “C-scan” is a two-dimensional, cross-sectional or“plan” view of the sample under examination, e.g., a two-dimensionalscan in the x-y plane. See, e.g., FIG. 1.

As used herein, the term “image segmentation” or “segmentation” refersto a digital method of dividing image data into regions that may consistof a pixel area that is homogeneous in terms of certain characteristics,or of an area that groups pixels corresponding to an object that isvisualized in the image. In this way, multiple layers or image fragmentsmay be created, for example, to represent tissue layers or regions of atissue that have similar characteristics. Accordingly, segmentationrefers to the process of partitioning a digital image into multipleregions (e.g., sets of pixels). In some embodiments, the goal ofsegmentation is to simplify and change the representation of an imageinto something that is more meaningful and easier to analyze. Imagesegmentation is typically used to locate objects and boundaries (lines,curves, etc.) in images. The result of image segmentation is a set ofregions that collectively cover the entire image, or a set of contoursextracted from the image. In some embodiments, the segments correspondto biological features (e.g., tissues, tissue layers, etc.). However,the technology is not limited to segments that correspond to biologicalfeatures and, in various embodiments, the segments correspond to anydivision of the image appropriate for the methods, technology, analysis,etc. desired by the user. Methods for finding and segmenting a desiredtissue layer or boundary surface are well-known in the art. See, e.g.,Ishikawa et al. (2005) “Macular Segmentation with Optical CoherenceTomography” Invest Ophthalmol Vis Sci 46; 2012, incorporated herein byreference in its entirety.

A “system” denotes a set of components, real or abstract, comprising awhole where each component interacts with or is related to at least oneother component within the whole.

As used herein, a “region of interest” refers to a region (e.g.,portion, sub-sample, sub-volume, etc.) of an image and/or of a sample(e.g., a tissue) that is assessed by the methods provided herein. Inparticular embodiments, the “region of interest” refers to a tissueabnormality, lesion, or other feature of a tissue that is subjected tothe metric analysis (e.g., measurement of an area; measurement of avolume) provided herein.

As used herein, an “increase” or a “decrease” refers to a detectable(e.g., measured) positive or negative change in the value of a variable(e.g., a volume) relative to a previously measured value of thevariable, relative to a pre-established value, and/or relative to avalue of a standard control. An increase is a positive change relativeto the previously measured value of the variable, the pre-establishedvalue, and/or the value of a standard control. Similarly, a decrease isa negative change relative to the previously measured value of thevariable, the pre-established value, and/or the value of a standardcontrol. Other terms indicating quantitative changes or differences,such as “more” or “less,” are used herein in the same fashion asdescribed above.

Description

Optical coherence tomography (OCT) is a method of using interferometryto determine the echo time delay and magnitude of backscattered lightreflected off an object of interest. OCT is similar in principle toultrasound, but in OCT light is used instead of sound and interferometryis used to determine the time delay of reflected light. The original OCTmethod, known as TD-OCT, encoded the location of each reflection in thetime information relating the position of a moving reference mirror tothe location of the reflection.

An advance in OCT was the use of light wavelengths instead of time delayto determine the spatial location of reflected light. Fourier transformanalysis is used to provide a technology based in the spectral domain(SD-OCT) rather than in the time domain (TD-OCT). SD-OCT acquires allinformation in a single axial scan through the tissue simultaneously byevaluating the frequency spectrum of the interference between thereflected light and a stationary reference mirror. See, e.g., Wojtkowskiet al. (2004) “Ophthalmic imaging by spectral optical coherencetomography” Am J Ophthalmol 138: 412-9; Wojtkowski et al. (2002) “Invivo human retinal imaging by Fourier domain optical coherencetomography” J Biomed Opt 7: 457-63; and Wojtkowski et al. (2003)“Real-time in vivo imaging by high-speed spectral optical coherencetomography” Opt Lett 28: 1745-47, each incorporated herein in itsentirety by reference.

SD-OCT is advantageous over TD-OCT because the interference pattern issplit by a grating into its frequency components and all of thesecomponents are simultaneously detected by a charge-coupled device (CCD),thus making it faster. Further, data are acquired without mechanicalmovement of a scanning mirror as in TD-OCT. The SD-OCT techniquesignificantly increases signal-to-noise ratio and increases the speed ofdata collection by a factor of 50 relative to TD-OCT. For example, aconventional time-domain OCT functions at 400 A-scan/s, while an SD-OCTsystem scans at 20,000 A-scan/s. Because of the increase in speed, asingle cross-sectional scan of 1000 A-scans can be captured, processed,streamed to disk, and displayed in 60 ms (or 1/42 of the time requiredfor a time-domain scan). Because of this speed, there is less movementof the subject during the SD-OCT scan and thus a more stable image isproduced with a significant decrease in artifact of the image. Alsobecause of this speed, a stack of 100 cross-sectional scans can beacquired in the time normally used to gather 6 low-resolutioncross-sectional scans on a time-domain system. The image stack can beprocessed to produce a three dimensional representation of structures(see Wojtkowski et al. (2005) “Three-dimensional retinal imaging withhigh-speed ultrahigh-resolution optical coherence tomography”Ophthalmology 112: 1734-46, incorporated herein by reference).

SD-OCT imaging thus frequently uses a series of scans. Focusing thelight beam to a point on the surface of the sample under test, andrecombining the reflected light with the reference will yield aninterferogram with sample information corresponding to a single A-scan(along the z-axis). Scanning of the sample can be accomplished by eitherscanning the light on the sample, or by moving the sample under test. Alinear scan will yield a two-dimensional data set corresponding to across-sectional image (e.g., in the x-z plane), whereas an area scanachieves a three-dimensional data set corresponding to a volumetricimage (e.g., a volume in the x-y-z space), also called full-field OCT.Accordingly, a stack of B-scans can undergo further analysis and producea three dimensional representation of structures.

Furthermore, it is possible to collapse three-dimensional OCT volumes(e.g., along a z-axis (e.g., along the depth axis)) to a two-dimensionalrepresentative image along any plane of a 3D volume using algorithms tocalculate a single representative pixel intensity for each line in theprojection. One technique of obtaining such an “en face” picture withoptical coherence tomograms is referred to as a summed voxel projection(SVP) (see, e.g., Jiao et al (2005) “Simultaneous acquisition ofsectional and fundus ophthalmic images with spectral-domain opticalcoherence tomography” Optics Express 13: 444-452, incorporated herein byreference).

Image registration and alignment is based on tissue structural features,e.g., to correct motion artifacts (see, e.g., Jorgensen et al (2007)“Enhancing the signal-to-noise ratio in ophthalmic optical coherencetomography by image registration-method and clinical examples” J BiomedOpt 12: 041208). For example, 3D data sets are presented with all pixelsin each given axial scan summed to produce an OCT fundus image, whichresembles a 2D photograph summing reflections from all tissue layers.The OCT fundus image can be used for image alignment or registrationbased on tissue features, such as blood vessel continuities ordiscontinuities. The 3D OCT can also be aligned or registered to afundus photograph acquired simultaneously or nearly so. Automated ormanual segmenting defines tissue layers in the SD-OCT data.

Because of the unique optically clear pathway through the eye, OCT hasbeen used for imaging disorders affecting the retina. In some currentuses, obtaining and processing each of a series of 500×500-pixel imagestakes on the order of seconds and the technology can now acquire 3D datasets comprising several hundred scans of 200×200×1024 pixels in 2seconds. In exemplary embodiments, this method is used to scan throughthe layers of a structured tissue sample such as the retina with veryhigh axial resolution (3 to 15 μm), providing images demonstrating 3Dstructure.

SD-OCT images show multiple tissue (e.g., retinal) layers of differentreflectivity. These tissue layers are typically segmented using acomputer algorithm and/or by manual tracing. When an abnormality occursin a tissue (e.g., in the retina (e.g., a “retinal lesion”)), thelayered structure of the tissue (e.g., retina) is altered, resulting ina thickening, thinning, or loss of tissue (e.g., retinal, RPE) layers atthe corresponding location, which are imaged by SD-OCT. In someembodiments, the lesion is present in the image as a protrusion in oneof the segmented features of the image. Thus, volumetric analysis oftissue abnormalities, lesions, etc. is desirable to evaluate, monitor,and treat the abnormalities, lesions, etc.

Examples of OCT display image technologies are provided, e.g., by U.S.Pat. No. 8,944,597, incorporated herein by reference. See also U.S. Pat.No. 8,913,793 (incorporated herein by reference in its entirety), whichrelates to display of OCT images in various ways, includingthree-dimensional surface renderings, topographical contour maps,contour maps, en-face color maps, and en-face grayscale maps.

Further, some embodiments related to retinal pathology provideclinicians with a cross-section of the pathology in the context of a mapof the retina. For example, some embodiments provide a cross-section ofa retinal abnormality presented in the context of a retinal thicknessmap. In some embodiments, two sequential scans of differing types (e.g.,resolutions) are performed and simultaneously displayed, preferably onthe same display. In some embodiments, the two display types areacquired using a single interaction with the user interface, say asingle click or a single voice command.

Paunescu et al. (“Reproducibility of nerve fiber thickness, macularthickness, and optic nerve head measurements using StratusOCT” InvestOphthalmol Vis Sci 45 (6) 1716-24, incorporated herein by reference inits entirety) describe methods of capturing a fundus image nearly“simultaneously” with the OCT, showing the location of the OCT beam onthe retina. “Simultaneity”, as used herein, simply means that datacollection happens quickly enough that the side-by-side display of thetwo types of data are sufficiently synchronized that they present twoviews of the same object and structure. U.S. Pat. App. Pub. No.2003/0199769 (incorporated herein by reference in its entirety), forexample, suggests taking a Scanning Laser Ophthalmoscope (SLO) imagepoint-by-point simultaneously with the OCT scan. This approach uses anadditional imaging system consisting of a beam splitter and the SLOdetector, and depends on hardware alignment between the OCT and SLOdetectors. For the purpose of providing a fast fundus image, a LineScanning Laser Ophthalmoscope (LSLO) is generally faster than the SLOand equally useful, as is the line-scan ophthalmoscope (LSO) of U.S.Patent Publication No. 2006/0228011, incorporated herein by reference inits entirety.

Various embodiments are related to visualization of images, e.g., toprovide output to a user and to convey results of image analysis methodsas described herein. For example, some embodiments provide informationuseful for live-time decisions and/or planning of clinical treatments,for analysis of previous clinical treatments (stents, drugs, genes,etc.), for similar purposes in preclinical studies, etc.

Automated segmentation results may be displayed in cross-sectional viewor longitudinal view or en face view. In addition, images may bedisplayed in a three-dimensional view or a “fly-through” view. Differentfeatures may be displayed using different shading relative to oneanother or as different colors.

Quantification results may be displayed in an image view and/or reportedin tables or text.

In some embodiments, surface and/or volume visualization techniques areused to provide views of the three-dimensional image data from any angleand, in some embodiments, with virtual lighting from any angle, in aninteractive fashion. In some embodiments, such volumes are digitallysliced along any plane or arbitrary surface to create a reformatted twodimensional view.

Software for visualization and analysis of biological image data includethose sold under the trade names of ParaView, ScanImage, μManager,MicroPilot, ImageJ, Vaa3D, ilastik (which includes machine learning,e.g., to aid a user in identifying image features), CellProfiler,CellExplorer, BrainExplorer, Zen (Zeiss), Amira (VSG), Imaris(Bitplane), ImagePro (Me diaCybernetics), Neurolucida (MBF Bioscience),LabVIEW (National Instruments), MATLAB (Mathworks), and Virtual Finger(see, e.g., Peng et al (2014) Nature Communications 5: 4342). See also,Walter et al (2010) Nature Methods 7: S26-S41; Eliceiri et al (2013)Nature Methods 9: 697; and Long (2012) PLoS Computational Biology 9:e1002519, each incorporated herein in its entirety. Further, in someembodiments the technology incorporates an image analysis library suchas VTK, ITK, OpenCV, or the Java ImgLib.

Methods

Provided herein are embodiments of methods for processing and analyzingOCT image data. In some embodiments, the methods provide one or moremeasurements (e.g., distance, area, and/or volume measurements; e.g.,measurements in one, two, or three dimensions, and, in some embodiments,measurements in one, two, or three dimensions as a function of time)Accordingly, in some embodiments the methods provide a technology tomonitor changes is the size, location, and/or shape of lesions of theretina, layers of the retina, subretinal tissue, and RPE. For example,particular embodiments relate to a method for determining the areaand/or volume of a region of interest within a biological tissue usingan image produced by optical coherence tomography. The method comprisesproducing, acquiring, analyzing, displaying, manipulating, etc.three-dimensional OCT data and producing, acquiring, analyzing,displaying, manipulating, etc. two-dimensional “fundus” OCT data. Forexample, the three-dimensional OCT data provide a three-dimensionalimage of the biological tissue comprising the region of interest and thetwo-dimensional OCT data are fundus image data of the biological tissuecomprising the region of interest.

In some preferred embodiments, the two-dimensional fundus data areassociated with (e.g., registered with, linked to, etc.) thethree-dimensional image of the biological tissue. In some embodiments,user interaction with the two-dimensional image data (e.g., analyzing,displaying, manipulating, etc. the two-dimensional image data) producesa linked, associated, coordinated interaction (e.g., analysis, display,manipulation, etc.) of the three-dimensional image data. For example, insome embodiments, methods comprise display of the two-dimensional fundusdata and user interaction with the display of the two-dimensional fundusdata. Then, in some embodiments, a user interacts with thetwo-dimensional fundus data—e.g., the user interacts with the display ofthe two-dimensional fundus data by use of an input device, e.g., a touchscreen, mouse, track ball, etc. to provide a boundary around the regionof interest and the user receives sensory feedback, e.g., the boundaryis displayed superimposed on the two-dimensional fundus image data asthe user interacts with the displayed image. Further, indication of theboundary around the region of interest in the two-dimensional fundusimage provides an associated, coordinated boundary around the region ofinterest in the three-dimensional image data. In this way, the user,“draws” the boundary around the region of interest using the technologyprovided herein, e.g., using a combination of the OCT image data (e.g.,the three-dimensional image data and associated two-dimensional fundusimage data), an output device (e.g., display), an input device (e.g., atouch screen), and a computer configured to calculate the area and/orvolume of a region of interest according to the methods and technologiesdescribed herein.

In some embodiments, user interaction with the three-dimensional OCTdata (e.g., analyzing, displaying, manipulating, etc. thethree-dimensional OCT data) produces a linked, associated, coordinatedinteraction (e.g., analysis, display, manipulation, etc.) of thetwo-dimensional fundus data. For example, in some embodiments, methodscomprise display of the three-dimensional OCT data and user interactionwith the display of the three-dimensional OCT data (e.g., examination ofone or more “slices” of the three-dimensional OCT data, by “fly-through”of the OCT data, or by otherwise examining the three-dimensional OCTdata on a display). Then, in some embodiments, a user interacts with thethree-dimensional OCT data—e.g., the user interacts with the display ofthe three-dimensional OCT data by use of an input device, e.g., a touchscreen, mouse, track ball, etc. to provide a boundary around the regionof interest and the user receives sensory feedback, e.g., the boundaryis displayed superimposed on the three-dimensional OCT data and/or onthe two-dimensional fundus image data as the user interacts with thedisplayed image. Further, indication of the boundary around the regionof interest in the three-dimensional OCT image provides an associated,coordinated boundary around the region of interest in thetwo-dimensional image data. In this way, the user, “draws” the boundaryaround the region of interest using the technology provided herein,e.g., using a combination of the OCT image data (e.g., thethree-dimensional image data and associated two-dimensional fundus imagedata), an output device (e.g., display), an input device (e.g., a touchscreen), and a computer configured to calculate the area and/or volumeof a region of interest according to the methods and technologiesdescribed herein.

In some embodiments, a user provides a continuous boundary around theregion of interest. In some embodiments a user provides a discontinuousboundary (e.g., a series of points, dots, lines, line segments (e.g.,straight line segments, curved line segments), etc.) marking some of theregion of interest (e.g., marking one or more locations of the edge ofthe region of interest). In some embodiments, a user provides points orportions of a boundary around a region of interest and an automatedimage processing algorithm completes the boundary using image analysisand the user-defined points or partial boundary to complete the boundary(e.g., using interpolation analysis to connect the user-providedportions of the boundary).

In embodiments of the technology in which the images are segmented, thetechnology is not limited by how the images are segmented. For example,various embodiments provide for the automated segmentation of the images(e.g., by computer algorithm that identifies image segments),semi-automated segmentation, or manual segmentation of the image (e.g.,by a user who identifies image segments). See also, U.S. Pat. No.8,811,745 (incorporated herein by reference), which describes systemsand methods for segmentation and identification of structured featuresin images (e.g., an ocular image showing layered structures or otherfeatures of the retina). Some embodiments further provide for automateddetection and identification (e.g., marking) of biological features inimages such as, e.g., blood vessels. See, e.g., U.S. Pat. No. 8,750,615(incorporated herein by reference in its entirety), which describes asystem and related methods for automatic or semi-automatic segmentationand quantification of blood vessel structure and physiology, includingsegmentation, quantification, and visualization of vessel walls,plaques, and macrophages.

The image processing technology provides in particular a method formeasuring a linear distance, an area, and/or a volume of a region ofinterest within a biological tissue using an image produced by opticalcoherence tomography. In an exemplary embodiment, an OCT apparatus(e.g., a SD-OCT apparatus) and a tissue are positioned for acquisitionof OCT data (e.g., OCT image data such as, e.g., SD-OCT image datacomprising three-dimensional image data and a fundus image). See, e.g.,FIG. 1 showing an OCT apparatus (“OCT”) and a sample (e.g., a tissue) ina schematic drawing. After acquiring OCT data (e.g., three dimensionalOCT image data), the data are segmented to produce an image showing thesegments (e.g., representing tissue layers and/or other features of thesample). For example, FIG. 2A (bottom panel) shows a projection ofthree-dimensional OCT data (e.g., an image as shown on a display suchas, e.g., a computer screen) in two dimensions (e.g., a cross-section ina plane parallel, effectively parallel, and/or substantially parallel tothe z-axis). The example OCT image in FIG. 2A (bottom panel) has beensegmented (see, e.g., upper and lower lines corresponding to a firstsegment and a second segment), e.g., to show tissue layers. Further, theexemplary OCT image shown in FIG. 2A (bottom panel) comprises a regionof interest as a protrusion in the upper segment. In exemplaryembodiments, such a protrusion may indicate abnormal tissue growth, alesion (a retinal lesion), central neovascularization (e.g., associatedwith macular degeneration) or other abnormal feature in a biologicaltissue. Also shown in FIG. 2A (upper panel) is an exemplary OCT fundusimage (e.g., as shown on a display such as, e.g., a computer screen) ina plane normal, effectively normal, and/or substantially normal to thez-axis (e.g., in the x-y plane). The exemplary fundus image shown inFIG. 2A (upper panel) shows the region of interest (FIG. 2A (upperpanel), black outlined shape).

According to embodiments of the technology provided herein, the imagesare analyzed to determine the area and/or volume of the region ofinterest (e.g., the protrusion shown in FIG. 2A (bottom panel)).

In some embodiments, the greatest linear dimension of the region ofinterest is determined (e.g., by examination of the fundus image and/orthe three dimensional OCT data (e.g., image)). See FIG. 2B (upperpanel), g. The greatest linear dimension is the greatest distance acrossthe region of interest. For example, the greatest linear dimension canbe determined by identifying the longest line segment having each of itstwo ends touching the perimeter of the region of interest. In someembodiments, the greatest linear dimension of the region of interest isprovided by a user. In particular, in some embodiments the fundus imageis provided to a user on a display and the user draws a line segmenthaving each of its two ends touching the perimeter of the region ofinterest using a computer and computer input device (e.g., mouse, touchscreen, light pen, etc.). As the user draws the line segment, the linesegment is provided on the fundus image of the region of interest on thedisplay. In some embodiments, a computer determines and provides thegreatest linear dimension of the region of interest (e.g., byidentifying the longest line segment having each of its two endstouching the perimeter of the region of interest). In some embodiments,the computer displays a line on a display showing the greatest lineardimension of the region of interest.

In some embodiments, a boundary is provided around the region ofinterest, e.g., an area enclosing the region of interest is identifiedin the fundus image. For example, in some embodiments a circle having adiameter (see, e.g., FIG. 2B (top panel), d) greater than or equal tothe greatest linear dimension g is provided to circumscribe the regionof interest. The boundary has an area A (see, e.g., FIG. 2B (top panel),grey region) and the region of interest is within the area A.

In embodiments in which the boundary is a circle, the area A=Π×(d/2)².

The technology is not limited in the shape of the boundary. The boundarymay be any shape (e.g., circle, ellipse, square, etc., or an irregularshape) enclosing the region of interest and having an area. See, e.g.,FIG. 2D-1 (showing a circle boundary) and FIG. 2D-2 (showing anirregular boundary). In some embodiments, a computer determines theboundary. In some embodiments, a user determines the boundary. Forexample, in some embodiments the fundus image is provided to a user on adisplay and the user draws a shape enclosing the region of interestusing a computer and computer input device (e.g., mouse, touch screen,light pen, etc.). As the user draws the boundary, the boundary isprovided on the fundus image of the region of interest on the display.In preferred embodiments, the area A of the boundary is determined bycomputer analysis, e.g., according to algorithms for determining thearea of shapes (e.g., irregular shapes).

Extension of the boundary substantially in the direction of the z-axis(e.g., through the sample (e.g., tissue)) defines a volume v in thethree dimensional OCT data (e.g., image). The volume v is defined by thefirst and second segments and by extension of the boundary through thesegments. FIG. 2B (bottom panel), grey region, shows a cross-section ofthe volume defined by the first segment, second segment, and theextended boundary. Computer analysis of the three dimensional data(e.g., image data) provides a volume v of the volume defined by thefirst segment, second segment, and the extended boundary.

The data are analyzed to determine a distance t (e.g., thickness)between the first segment and the second segment in the direction of thez-axis. In particular embodiments, t is the average distance between thefirst segment and the second segment measured along the perimeter of theboundary. In alternative embodiments, the distance t may also be themaximum distance between the first segment and the second segmentmeasured along the perimeter of the boundary, the minimum distancebetween the first segment and the second segment measured along theperimeter of the boundary, and/or any other distance calculated betweenthe first segment and the second segment measured along the perimeter ofthe boundary. Average distance may be calculated using an averagecalculated in a sliding window moved along the perimeter of theboundary. The distance t provides a measurement for the normal distancebetween the first segment and the second segment in a normal sample(e.g., a normal tissue), e.g., a sample that does not comprise abnormalgrowth, does not comprise a lesion, etc. As such, preferred embodimentsare those in which the boundary is provided in a region of the data(e.g., images) corresponding to healthy, normal sample (e.g., healthy,normal tissue), e.g., healthy, normal, etc. relative to the region ofinterest, which corresponds to abnormal sample (e.g., abnormal tissuecomprising a feature such as a lesion).

The technology provides methods for calculating the area A and/or thevolume V of a region of interest (e.g., an abnormality, lesion, etc.).Thus, in some embodiments, the area A defined by the boundary and thedistance t are used to calculate a volume n. The volume n is subtractedfrom the volume v determined above to provide the volume V of the regionof interest (e.g., abnormality, lesion, etc.). Accordingly, the volume nis calculated as the product of the area A of the boundary and thethickness t, as determined above. FIG. 2C (lower panel) shows a volume nin cross-sectional view (white rectangle). The volume n has a heightthat is the distance t. The top and bottom of the volume n each have anarea A. Accordingly, the volume V of the region of interest (FIG. 2C(lower panel), grey region) is calculated by subtracting the volume n(FIG. 2C (lower panel), white region) from the volume v (FIG. 2B (lowerpanel), grey region).

While certain embodiments are described above using a boundary that is acircle, the technology comprises use of a boundary of any shape (e.g.,circle, ellipse, square, etc., or an irregular shape) enclosing theregion of interest and having an area A. For example, FIG. 2D-1 and FIG.2D-2 show a volume n having a top and bottom that are circles havingarea A and height t. FIG. 2D-3 and FIG. 2D-4 show a volume n having atop and bottom that are an irregular shape having area A and height t.

In some embodiments, the area A is determined from examining thethree-dimensional OCT image to localize the margins of the area on thetwo-dimensional image. For example, in some embodiments, area A iscalculated from the interpolation of points on the edges of the areausing a three-dimensional image that is registered with thetwo-dimensional image.

Accordingly, the technology provides a general method for determiningthe area A and/or the volume V of a region of interest in OCT data,e.g., comprising the steps of acquiring OCT data, determining the volumev (e.g., defined by the first and second segments and by the extensionof the boundary through the segments), calculating the volume n (e.g.,as the product of the area A of the boundary and the distance t), andsubtracting n from v.

Systems

Some embodiments of the technology provide systems determining the areaand/or the volume of a region of interest in OCT data (e.g., in OCT dataacquired from a biological tissue, e.g., OCT image of a biologicaltissue such as a retina). Systems according to the technology comprise,e.g., an OCT apparatus (e.g., a SD-OCT apparatus), a computer, andsoftware to instruct a computer to perform a method as described herein.Some embodiments further comprise a display (e.g., to provide threedimensional OCT data (e.g., three dimensional OCT images) and/or twodimensional OCT data (e.g., a fundus image) to a user) and an inputdevice (e.g., for a user to provide information to the computer (e.g.,to provide a boundary enclosing a region of interest).

For example, in some embodiments, computer-based analysis is used tocalculate the area A of the boundary, determine the distance t betweenthe first segment and the second segment, calculate the volume v (e.g.,defined by the first segment, second segment, and the boundary extendedthrough the segments), and the volume n (e.g., product of area A anddistance t), and volume V (volume of the region of interest). In someembodiments, one or more of these calculations use data provided by auser and/or data acquired by the computer.

For instance, some embodiments comprise a computer system upon whichembodiments of the present technology may be implemented. In variousembodiments, a computer system includes a bus or other communicationmechanism for communicating information and a processor coupled with thebus for processing information. In various embodiments, the computersystem includes a memory, which can be a random access memory (RAM) orother dynamic storage device, coupled to the bus, and instructions to beexecuted by the processor. Memory also can be used for storing temporaryvariables or other intermediate information during execution ofinstructions to be executed by the processor. In various embodiments,the computer system can further include a read only memory (ROM) orother static storage device coupled to the bus for storing staticinformation and instructions for the processor. A storage device, suchas a magnetic disk or optical disk, can be provided and coupled to thebus for storing information and instructions.

In various embodiments, the computer system is coupled via the bus to adisplay, such as a cathode ray tube (CRT) or a liquid crystal display(LCD), for displaying information to a computer user (e.g., threedimensional OCT images and/or two dimensional OCT images such as afundus image).

An input device, including alphanumeric and other keys, can be coupledto the bus for communicating information and command selections to theprocessor. Another type of user input device is a cursor control, suchas a mouse, a trackball, a light pen, a touch screen, or cursordirection keys, for communicating direction information and commandselections to the processor and for controlling cursor movement on thedisplay (e.g., to draw shapes, lines, etc. to show on the computerdisplay). This input device typically has two degrees of freedom in twoaxes, a first axis (e.g., x) and a second axis (e.g., y), that allowsthe device to specify positions in a plane. These x and y axes are notnecessarily coincident with the x and y axes shown in FIG. 1 (e.g., withrespect to the sample and images).

A computer system can perform embodiments of the present technology.Consistent with certain implementations of the present technology,results can be provided by the computer system in response to theprocessor executing one or more sequences of one or more instructionscontained in the memory. Such instructions can be read into the memoryfrom another computer-readable medium, such as a storage device.Execution of the sequences of instructions contained in the memory cancause the processor to perform the methods described herein.Alternatively, hard-wired circuitry can be used in place of or incombination with software instructions to implement the presentteachings. Thus, implementations of the present technology are notlimited to any specific combination of hardware circuitry and software.

The term “computer-readable medium” as used herein refers to any mediumor media that participates in providing instructions to the processorfor execution. Such a medium can take many forms, including but notlimited to, non-volatile media, volatile media, and transmission media.Examples of non-volatile media can include, but are not limited to,optical or magnetic disks, such as a storage device. Examples ofvolatile media can include, but are not limited to, dynamic memory.Examples of transmission media can include, but are not limited to,coaxial cables, copper wire, and fiber optics, including the wires thatcomprise the bus.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, any other optical medium, punch cards, paper tape, anyother physical medium with patterns of holes, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, or any other tangiblemedium from which a computer can read.

Various forms of computer readable media can be involved in carrying oneor more sequences of one or more instructions to the processor forexecution. For example, the instructions can initially be carried on themagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over anetwork connection (e.g., a LAN, a WAN, the internet, a telephone line).A local computer system can receive the data and transmit it to the bus.The bus can carry the data to the memory, from which the processorretrieves and executes the instructions. The instructions received bythe memory may optionally be stored on a storage device either before orafter execution by the processor.

In accordance with various embodiments, instructions configured to beexecuted by a processor to perform a method are stored on acomputer-readable medium. The computer-readable medium can be a devicethat stores digital information. For example, a computer-readable mediumincludes a compact disc read-only memory (CD-ROM) as is known in the artfor storing software. The computer-readable medium is accessed by aprocessor suitable for executing instructions configured to be executed.

In accordance with such a computer system, some embodiments of thetechnology provided herein further comprise functionalities forcollecting, storing, and/or analyzing data (e.g., OCT images, e.g.,three dimensional OCT images, two dimensional OCT images). For example,some embodiments contemplate a system that comprises a processor, amemory, and/or a database for, e.g., storing and executing instructions,analyzing image data, performing calculations using the data,transforming the data, and storing the data. It some embodiments, analgorithm applies a model for calculating (e.g., approximating) an areaand/or a volume in the image data. Some embodiments provide for theresizing, cropping, flattening, or other manipulation of image data.Particular embodiments provide a database to organize, search, process,analyze, share and visualize image data and image metadata.

In some embodiments, area and/or volume data (e.g., comprisinginformation relating to the area A and/or volume V of a region ofinterest for a subject) are stored (e.g., associated with a time atwhich the area A and/or volume V is determined and/or associated withthe particular subject. For example, volume data (e.g., comprisinginformation relating to the area A and/or volume V of a region ofinterest for a subject) are acquired at more than one time (e.g., over aperiod of days, weeks, months, years, or decade) and an area A (e.g.,A₁) and/or a volume V (e.g., V₁) acquired at one time is compared to anarea A (e.g., A₂) and/or a volume V (e.g., V₂) acquired at another time.In some embodiments, the difference in the two values of A (e.g., A₂−A₁)and/or V (e.g., V₂−V₁) is used to inform a treatment of the subject. Forexample, in some embodiments the magnitude of the area A₁ and/or volumeV₁ acquired at one time is used to determine a treatment, dosage,pharmaceutical administration, medical intervention (e.g., surgery),etc. Then, determining the area A₂ and/or volume V₂ at a later timeprovides an indication of the effectiveness of the treatment, e.g., insome embodiments an A₂ and/or a V₂ that is less than A₁ and/or V₁ forthe region of interest indicates that the treatment was effective.

Many diagnostics involve determining the presence of, size of, locationof, etc. a region of interest in a sample. Thus, in some embodiments, anequation comprising variables representing the presence of, size of,location of, etc. a region of interest in a sample produces a value thatfinds use in making a diagnosis or assessing the presence or qualitiesof a region of interest. As such, in some embodiments this value ispresented by a device, e.g., by an indicator related to the result(e.g., an LED, an icon on a display, a sound, or the like). In someembodiments, a device stores the value, transmits the value, or uses thevalue for additional calculations.

Thus, in some embodiments, the present technology provides the furtherbenefit that a clinician, who is not likely to be trained in imageanalysis, pathology, and/or the biology of particular tissues need notunderstand the raw data. The data are presented directly to theclinician in its most useful form. The clinician is then able to utilizethe information to optimize the care of a subject. The presenttechnology contemplates any method capable of receiving, processing, andtransmitting the information to and from laboratories conducting theassays, information providers, medical personal, and/or subjects. Forexample, in some embodiments of the present technology, data areacquired from analyzing a subject's tissue and the data are submitted toan analysis service (e.g., a clinical lab at a medical facility, atissue profiling business, etc.), located in any part of the world(e.g., in a country different than the country where the subject residesor where the information is ultimately used). For example, the subjectmay visit a medical center to be tested and to have data sent to theprofiling center. Where the data comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., data transmitted to a computerof the profiling center using electronic communication systems). Oncereceived by the profiling service, the data are processed and a profileis produced that is specific for the diagnostic or prognosticinformation desired for the subject. The profile data are then preparedin a format suitable for interpretation by a treating clinician. Forexample, rather than providing raw image data, the prepared format mayrepresent a diagnosis or risk assessment for the subject, along withrecommendations for particular treatment options. The data may bedisplayed to the clinician by any suitable method. For example, in someembodiments, the profiling service generates a report that can beprinted for the clinician (e.g., at the point of care) or displayed tothe clinician on a computer display. In some embodiments, theinformation is first analyzed at the point of care or at a regionalfacility. The raw data are then sent to a central processing facilityfor further analysis and/or to convert the raw data to informationuseful for a clinician or patient. The central processing facilityprovides the advantage of privacy (all data are stored in a centralfacility with uniform security protocols), speed, and uniformity of dataanalysis. The central processing facility can then control the fate ofthe data following treatment of the subject. For example, using anelectronic communication system, the central facility can provide datato the clinician, the subject, or researchers. In some embodiments, thesubject is able to access the data using the electronic communicationsystem. The subject may chose further intervention or counseling basedon the results. In some embodiments, the data are used for research use.For example, the data may be used to further optimize the inclusion orelimination of markers as useful indicators of a particular conditionassociated with a disease.

Applications

OCT is widely used, for example, to obtain high-resolution images of theanterior segment of the eye and the retina. As such, the technique findsuse, for example, in assessing axonal integrity in diseases such as,e.g., multiple sclerosis, other neurodegenerative diseases, andglaucoma. OCT finds use for monitoring the progression of glaucoma andto image coronary arteries to detect lipid-rich plaques. In an exemplaryuse, the technology finds use in measuring retinal thickness. Retinalthickness may be abnormally large in cases of retinal edema or tractionby membranes in the vitreous humor. On the other hand, the retina and/orRFE may appear thin or absent in cases of atrophic degeneration,chorioretinitis, or trauma to the retina. Meanwhile, changes in retinalthickness may be localized or extend over large areas. In certain cases,the overall contour of the retina may become abnormal. For example,pronounced myopia, particularly due to posterior staphylomas, may createa highly concave retina. Retina layers overlying regions of RPE atrophymay become markedly thinned or lost. Detachment of the retinal pigmentepithelium (RPE), subretinal cysts, or subretinal tumors may produce arelative convexity of the retina. Therefore, mapping the retina contouror retinal thickness makes it possible to determine the extent andseverity of such conditions and to monitor progress of treatment.

In addition, the technique finds use in imaging brain tissue in vivo,e.g., using OCT to produce detailed images of mice brains through atransparent zirconia window implanted in the skull. OCT finds use toidentify root canals in teeth (e.g., canal in the maxillary molar).Also, OCT finds use in interventional cardiology to diagnose coronaryartery disease.

Furthermore, OCT finds use in industrial applications, such as innon-destructive testing (NDT), material thickness measurements, and forexamining thin silicon wafers and compound semiconductor wafers (e.g.,to make thickness measurements, surface roughness characterization,surface imaging, cross-section imaging, and volume loss measurements).OCT systems with feedback can be used to control manufacturingprocesses. OCT finds use in the pharmaceutical industry to control thecoating of tablets.

In some embodiments, the technology finds use in metric analysis of aCNV lesion complex and/or a region of RPE loss, e.g., as associated withmacular degeneration, in OCT (e.g., SD-OCT) images (see, e.g., Examples2 and 3).

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EXAMPLES Prophetic Example 1

In some embodiments, the technology finds use in diagnosing and treatinga patient. For example, the technology aids a physician who determinesthat a patient has a subretinal choroidal neovascular membrane withsubretinal fluid developing in the macula of an eye. A volumetric rasterOCT scan of a 6 mm×6 mm region of the central macula is obtained (e.g.,using default settings) to capture the 3D image. The OCT scan isregistered to a retinal angiographic image obtained during the samevisit. The physician determines the boundaries of the lesion in thefundus angiogram image. Using a computer mouse, the user defines aregion that includes the lesion but extends beyond it into retinaltissue that appears normal, thereby defining the area A for analysis.The defined region happens to be irregular in shape (e.g., not perfectlycircular). The segmentation algorithm is run, which segments theinternal limiting membrane layer and the retinal pigment epitheliumlayer of the retina. The volume of the defined region of interest iscalculated. From this the volume of the abnormality, V₁, is calculatedby the software. This volume V₁ is 1.5 mm³. At this first visit, thepatient is given a drug treatment to treat the lesion. At a secondvisit, the scan and angiogram studies are repeated on the patient's eyeand the data are registered with software. Again, the physicianidentifies the region of interest and draws on the angiogram image theregion of interest that circumscribes the lesion and some normal retina,which is not circular. After the segmentation algorithm is run, a secondvolume from the second visit is obtained. From this, V₂ is calculated.V₂ is determined to be 0.75 mm³. The ratio V₂/V₁ is 0.5. The physiciandetermines that the treatment has lessened the volume of the abnormalityby 50%, indicating a treatment effect. The physician plans to continuetreatment with administration of the same drug at the second visit dueto a good initial response to treatment.

Example 2 Metric Analysis of a CNV Lesion

Quantitative analysis of OCT data has been used in clinical trialstargeting wet AMD in patients. In one class of treatments comprisingadministration of anti-VEGF agents (e.g., Lucentis, Eylea), metricevaluation of retinal thickness is used to monitor subretinal fluidaccumulation. In addition, combination therapies targeting VEGF and PDGFfind use in treatment of patients. In these treatments, metricassessment (e.g., measurement of the volume and/or area) of CNV is usedto monitor the effectiveness of the PDGF treatment. Accordingly, thetechnology described herein finds use in the quantitative analysis(e.g., metric analysis (e.g., determination of volume and/or area)) ofCNV size based on SD-OCT.

In an exemplary application of embodiments of the technology, an SD-OCTscan and an associated fundus image registered pixel-to-pixel to theSD-OCT data are provided. In some embodiments, the technology is basedon the use of SD-OCT data only, but an improved technology is providedby use of SD-OCT data and an associated fundus image. For example,providing both OCT data and a registered fundus image improve useranalysis and grading of the tissues and lesions in the patient. In someembodiments, the SD-OCT and fundus image are displayed on a display sideby side, e.g., in a split view mode, e.g., as provided by a softwareimplementation of the technology provided herein (see, e.g., FIG. 3A andFIG. 3B). In this view mode, the long horizontal white line in thefundus image of FIG. 3A marks the plane view of the OCT data displayedin FIG. 3B; and the location of the vertical white tick mark on thefundus image (FIG. 3A) is correlated to the location of the two verticalwhite tick marks on the OCT image (FIG. 3B).

FIG. 3A shows a fundus image and FIG. 3B shows an associated view of OCTdata. The fundus image and OCT scan show a CNV complex, which occupiesapproximately left three-quarters of the OCT image field in FIG. 3B(e.g., to the left of the white tick marks). The view of the OCT datashows thick, multi-layers of reflective materials that are packedtogether. The retina appears nearly normal in the right quarter of theOCT image (e.g., to the right of the white tick marks), where retinalpigment epithelium is visible and flat. The retinal pigment epitheliumappears to be nearing a normal state at the left edge of the OCT dataview shown. The left edge of the OCT image and the vertical tick marksin the OCT image mark the edges of the CNV lesion, e.g., in someembodiments a user marks the edge (e.g., boundary) of the CNV lesion andin some embodiments a method implemented in computer software marks theedge (e.g., boundary) of the CNV lesion.

Accordingly, using embodiments of the technology provided herein, a userexplores the OCT (e.g., SD-OCT) scan and/or fundus image to locate theedge of the CNV lesion. As the user evaluates the image(s) andidentifies the edge of the CNV lesion, the user marks the edge of thelesion on the fundus photo. Then, in some embodiments, after exploringthe OCT (e.g., SD-OCT) data set, the user identifies (e.g., traces,marks) the area encompassing the extent of the CNV lesion.Alternatively, in some embodiments, software automatically determinesand indicates the area encompassing the extent of the CNV lesion, e.g.,in some embodiments software uses image analysis and one or more points,dots, line segments, etc. provided by the user identifying the edge ofthe lesion. See, e.g., FIG. 4A and FIG. 5A showing the areas marked onfundus images and the views of the registered OCT data in FIG. 4B andFIG. 5B. FIG. 4A and FIG. 4B are image data of a patient's retinaacquired at a time point; FIG. 5A and FIG. 5B are image data of the sameregion of the same patient's retina at another (e.g., later) time point.The long horizontal white lines in the fundus images of FIG. 4A and FIG.5A marks the plane view of the OCT data displayed in FIG. 4B and FIG.5B, respectively; and the location of the vertical white tick mark onthe fundus image (FIG. 4A and FIG. 5A) is correlated to the location ofthe two vertical white tick marks on the OCT images (FIG. 4B and FIG.5B).

The area of the region of interest is then derived automaticallyaccording to the technology provided herein (e.g., by an algorithm todetermine the area of the region of interest defined by the lineencompassing the region of interest). After segmenting the image data,in some embodiments, the volume of the CNV lesion complex is calculated(e.g., by calculating the volume v within the boundary of area A andbetween the first segment and the second segment; calculating theaverage thickness t between the first segment and the second segmentalong the boundary (e.g., along the perimeter of area A); andcalculating the volume V of the region of interest using V=v−(t×A)).

In an exemplary use of the technology, a subject is enrolled in atreatment course and monitored by OCT imaging. At a visit, the area of aCNV lesion is 11.8 mm² or 4.64 disc area (see, e.g., FIG. 4A showing thesize of a lesion prior to a treatment). At a later visit of the samesubject, the area of the CNV lesion is reduced to 8.17 mm² or 3.21 discarea (see, e.g., FIG. 5A showing the size of the same region of the samepatient after treatment). The reduction in the area of the region ofinterest (e.g., the smaller area of the region of interest in FIG. 5Arelative to the area of the region of interest in FIG. 4A) indicatesthat the treatment is effective.

Example 3 Metric Analysis of a Region of RPE Loss

The technology finds use in managing the care and treatment of a patienthaving AMD, e.g., to monitor vision defects and associated lesions ofthe retina and/or RPE. For example, during examination of the patient,OCT data are obtained from the patient's eye. The data show a complexregion of RPE loss (see, e.g., FIG. 6A showing a B scan and an en faceinfrared image). A user scrolls through the stacked B scans in the 3Dimage to mark the border of the atrophy, e.g., because the structure ofinterest is not visible on, or not definitely located in, the en faceimage (FIG. 6B, showing the region of atrophy partially defined, usingthe boundary as the location where the layer of the external limitingmembrane of the retina is lost). Finally, the boundary within the areaof RPE loss is found (FIG. 6C, showing the completed circumscribedregion of RPE loss). The region is calculated to have an area of 4.75mm² and the distance of the nearest border of RPE lost to the fovealcenter is 150 microns.

Other useful metrics are provided by and/or calculated from parametersassociated with the boundaries of regions of interest as defined withthis methodology. For instance, metrics defining the shape of a lesionobtained from measurements described herein (e.g., from a measurement ofthe perimeter of the boundary of area A) have prognostic value in someembodiments of the technology (see, e.g., Domalpally (2013) “CircularityIndex as a Risk Factor for the Progression of Geographic Atrophy”Ophthalmology 120(12): 2666-71).

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the following claims.

I claim:
 1. A method of treating a subject having a tissue abnormality,the method comprising: a) acquiring an area and/or a volume of thetissue abnormality by: i) acquiring three-dimensional OCT datacomprising at least a first segment and a second segment comprising thetissue abnormality; ii) acquiring two-dimensional image data comprisingthe tissue abnormality, wherein the tissue abnormality in thethree-dimensional OCT data corresponds to the tissue abnormality in thetwo-dimensional data; iii) drawing a boundary around the tissueabnormality in the two-dimensional image data, the boundary enclosing anarea A, wherein the two-dimensional image data and three-dimensional OCTdata are registered via a computer processor so that the tissueabnormality is identified in the three-dimensional OCT data by theboundary drawn on the two-dimensional image and displayed on a computerscreen; iv) calculating the volume v within the boundary and between thefirst segment and the second segment; v) calculating the averagethickness t between the first segment and the second segment along theboundary; and vi) calculating the volume of the region of interestV=v−(t×A); and b) administering a treatment to the subject based on thearea and/or the volume of the tissue abnormality.
 2. The method of claim1 wherein the patient has macular degeneration and the tissueabnormality is a central neovascularization lesion complex.
 3. Themethod of claim 1 wherein the treatment comprises administering to thesubject an agent targeting vascular endothelial growth factor and/or anagent targeting platelet derived growth factor.
 4. The method of claim 1wherein the boundary is a circle, ellipse, polygon, or irregular shape.5. The method of claim 1 further comprising determining a greatestlinear distance across the tissue abnormality in the two-dimensionalimage data.
 6. The method of claim 1 wherein the boundary is acontinuous boundary or a discontinuous boundary comprising one or morepoints, dots, straight line segments, or curved line segments markingthe edges of the tissue abnormality.
 7. The method of claim 1 whereinthe three-dimensional OCT data is a three-dimensional OCT image.
 8. Themethod of claim 1 wherein the two-dimensional image data is OCT fundusdata, a two-dimensional image produced by integrating three-dimensionalOCT data, a cross-sectional slice of three-dimensional OCT data, or aphotograph.
 9. The method of claim 1 wherein the area A is calculated byinterpolating between a plurality of points indicating the boundary ofthe tissue abnormality.
 10. The method of claim 1 wherein a user drawsthe boundary around the region of interest or draws a plurality ofpoints indicating the boundary around the tissue abnormality.
 11. Themethod of claim 1 wherein a user draws the boundary around the region ofinterest or draws a plurality of points indicating the boundary aroundthe tissue abnormality using a computer input device.
 12. The method ofclaim 1 wherein a user draws the boundary around the tissue abnormalityor draws a plurality of points indicating the boundary around the tissueabnormality using a mouse, touchscreen, trackball, joystick, trackpad,stylus, or light pen.
 13. The method of claim 1 wherein automated imageprocessing draws the boundary around the tissue abnormality.
 14. Themethod of claim 1 wherein the boundary is provided on normal biologicaltissue.
 15. The method of claim 1 wherein the tissue abnormality is alesion in the biological tissue.
 16. The method of claim 1 wherein thebiological tissue is retinal tissue and the tissue abnormality is aretinal lesion.
 17. A method of identifying that a treatment of asubject having a tissue abnormality is successful, the methodcomprising: a) calculating a first area and/or a first volume of thetissue abnormality by: i) acquiring three-dimensional OCT datacomprising at least a first segment and a second segment comprising thetissue abnormality; ii) acquiring two-dimensional image data comprisingthe tissue abnormality, wherein the tissue abnormality in thethree-dimensional OCT data corresponds to the tissue abnormality in thetwo-dimensional data; iii) drawing a boundary around the tissueabnormality in the two-dimensional image data, the boundary enclosing anarea A, wherein the two-dimensional image data and three-dimensional OCTdata are registered via a computer processor so that the tissueabnormality is identified in the three-dimensional OCT data by theboundary drawn on the two-dimensional image and displayed on a computerscreen; iv) calculating the volume v within the boundary and between thefirst segment and the second segment; v) calculating the averagethickness t between the first segment and the second segment along theboundary; vi) calculating the volume of the region of interestV=v−(t×A); b) administering a treatment to the subject; c) calculating asecond area and/or a second volume of the tissue abnormality byrepeating step (a); and d) identifying the treatment of the patient aseffective when the second area and/or the second volume of the tissueabnormality is less than the first area and/or the first volume of thetissue abnormality.
 18. The method of claim 17 wherein the patient hasmacular degeneration and the tissue abnormality is a centralneovascularization lesion complex.
 19. The method of claim 17 whereinthe treatment comprises administering to the subject an agent targetingvascular endothelial growth factor and/or an agent targeting plateletderived growth factor.
 20. The method of claim 17 wherein the boundaryis a circle, ellipse, polygon, or irregular shape.
 21. The method ofclaim 17 further comprising determining a greatest linear distanceacross the tissue abnormality in the two-dimensional image data.
 22. Themethod of claim 17 wherein the boundary is a continuous boundary or adiscontinuous boundary comprising one or more points, dots, straightline segments, or curved line segments marking the edges of the tissueabnormality.
 23. The method of claim 17 wherein the three-dimensionalOCT data is a three-dimensional OCT image.
 24. The method of claim 17wherein the two-dimensional image data is OCT fundus data, atwo-dimensional image produced by integrating three-dimensional OCTdata, a cross-sectional slice of three-dimensional OCT data, or aphotograph.
 25. The method of claim 17 wherein the area A is calculatedby interpolating between a plurality of points indicating the boundaryof the tissue abnormality.
 26. The method of claim 17 wherein a userdraws the boundary around the region of interest or draws a plurality ofpoints indicating the boundary around the tissue abnormality.
 27. Themethod of claim 17 wherein a user draws the boundary around the regionof interest or draws a plurality of points indicating the boundaryaround the tissue abnormality using a computer input device.
 28. Themethod of claim 17 wherein a user draws the boundary around the tissueabnormality or draws a plurality of points indicating the boundaryaround the tissue abnormality using a mouse, touchscreen, trackball,joystick, trackpad, stylus, or light pen.
 29. The method of claim 17wherein automated image processing draws the boundary around the tissueabnormality.
 30. The method of claim 17 wherein the boundary is providedon normal biological tissue.
 31. The method of claim 17 wherein thetissue abnormality is a lesion in the biological tissue.
 32. The methodof claim 17 wherein the biological tissue is retinal tissue and thetissue abnormality is a retinal lesion.